HID EVOlution – qPCR/STR Setup System · USER BULLETIN | SEPTEMBER 2010 HID EVOlution™ –...

USER BULLETIN | SEPTEMBER 2010

HID EVOlution™ – qPCR/STR Setup System

SUBJECT: Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter Software

Purpose of this user bulletin

The Quantifiler® Duo DNA Quantification Kit (Quantifiler® Duo kit) is now supported for automated qPCR reaction setup on the HID EVOlution™ – qPCR/STR Setup System (qPCR/STR system). This user bulletin:

• Provides Quantifiler Duo kit-specific instructions to supplement the procedures in the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide (PN 4426903).

• Describes the validation and verification studies of quantitative PCR (qPCR) setup on the qPCR/STR system using the new Quantifiler Duo scripts and the Quantifiler Duo Converter Software v1.0.

In this user bulletin

This user bulletin covers:

■ Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

■ Validation of Quantifiler® Duo Kit on HID EVOlution™ – qPCR/STR Setup System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Prerequisites

This user bulletin assumes that:

• You have access to the following documents: – HID EVOlution™ – qPCR/STR Setup System Getting Started Guide (PN 4426903)– Quantifiler® Duo DNA Quantification Kit User’s Guide (PN 4391294)– Tecan HID EVOlution™ - qPCR/STR Setup System Application Manual– Tecan Freedom EVO® Operating Manual

• You have referred to the manufacturer’s instrument documentation for important safety information related to the use of the Tecan Freedom EVO® instrument.

SECTION 1

2 Quantifiler® Duo DNA Quantification Kit User Bulletin

Section 1 Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup System

This section covers:

■ Workflow for the Quantifiler® Duo DNA Quantification Kit. . . . . . . . . . . . . . . . . . 3

■ One-time tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Download and install Quantifiler® Duo scripts and Converter software. . . . . . . . 4Create detectors for the 7500 Real-Time PCR System . . . . . . . . . . . . . . . . . . . . . . . . 5

■ Prepare qPCR reagents and labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Determine required reagent volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Place reagents in the qPCR reagent block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Set up the samples, reagents, and plasticware on the worktable . . . . . . . . . . . . . . 10

■ Run automated qPCR reaction setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ Perform qPCR amplification and review results . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Convert and transfer the 7500 setup file. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Import the converted 7500 setup file and perform qPCR . . . . . . . . . . . . . . . . . . . . 12Analyze and review the qPCR results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Export and convert the 7500 results files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ STR PCR reaction setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Import the converted 7500 results files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3Quantifiler® Duo DNA Quantification Kit User Bulletin

Section 1 Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup SystemWorkflow for the Quantifiler® Duo DNA Quantification Kit

Workflow for the Quantifiler® Duo DNA Quantification Kit

To use the Quantifiler® Duo kit on the qPCR/STR system, follow the instructions in the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide (PN 4426903) with the added and modified procedures listed in Table 1 and explained on pages 4 through 15 of this user bulletin.

Note: Figure 1 on page 5 illustrates the files imported and exported throughout the automated HID workflow.

Table 1 Workflow for using the Quantifiler® Duo kit on the qPCR/STR system.

To perform this workflow step... Follow the procedures in...

One-time tasks

Perform one-time tasks a. Download and install Quantifiler® Duo scripts and Converter software on page 4.

b. Create detectors for the 7500 Real-Time PCR System on page 5.

c. If you have not already done so, perform the other one-time tasks described in the Getting Started Guide (GSG)‡ Chapter 2, “Pre-Run Procedures”.

‡ HID EVOlution™ – qPCR/STR Setup System Getting Started Guide (PN 4426903).

qPCR reaction setup and qPCR

1. Perform routine maintenance and other pre-run procedures

GSG Chapter 2, “Pre-Run Procedures”.

2. Prepare qPCR reagents and labware a. Determine required reagent volumes on page 6.

b. Place reagents in the qPCR reagent block on page 8.

c. Set up the samples, reagents, and plasticware on the worktable on page 10.

3. Run automated qPCR reaction setup GSG Chapter 4, “Run Automated qPCR Setup,” except that you use the scripts on page 11.

4. Perform qPCR and review results a. Convert and transfer the 7500 setup file on page 11.

b. Import the converted 7500 setup file and perform qPCR§ on page 12.

§ The converted 7500 setup file is imported as a SDS plate record.

c. Analyze and review the qPCR results on page 13.

d. Export and convert the 7500 results files on page 14.

STR PCR reaction setup

1. Prepare STR PCR reagents and plasticware GSG Chapter 6, “Prepare STR PCR Reagents and Labware”.

2. Run automated STR PCR reaction setup GSG Chapter 7, “Run Automated STR PCR Setup” except that you use the instructions for importing qPCR results on page 15.


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwareOne-time tasks

One-time tasks

Download and install Quantifiler® Duo scripts and Converter software

IMPORTANT! To use the Quantifiler® Duo kit on the qPCR/STR system, you must download and install the:• HID EVOlution™ - Quantifiler® Duo Converter Software v1.0• HID EVOlution™ - Quantifiler® Duo Scripts

About the Quantifiler® Duo kit Converter Software

The HID EVOlution™ - Quantifiler® Duo Converter Software ensures that the Quantifiler Duo assay sample data is handled correctly by the SDS and qPCR/STR system software. Use the Converter to:

• Convert the 7500 setup file before you import the file into the SDS software• Convert the 7500 results file before you import the file(s) into the qPCR/STR

system software

Figure 1 on page 5 illustrates the files imported and exported throughout the automated HID workflow. The Quantifiler Duo scripts, Converter software, and converted files are flagged with “*”.

Download and install software and scripts

1. Go to www.tecan.com/HID.

2. Click Download on the right side of the page under Product Details.

3. Download the Quantifiler® Duo scripts and Converter software.

4. Unzip the downloaded files, then follow the instructions included with the download to install the scripts and Converter.

Note: The scripts must be installed on the qPCR/STR system computer. The Converter software is stand-alone software (not part of the Freedom EVOware® or SDS software). For ease of use, we recommend that you install the Converter software on the qPCR/STR system computer.

Note: The downloaded files also include instructions for using the Converter: HID EVOlution™ - qPCR/STR Setup System, HID EVOlution™ - Combination System, Quantifiler® Duo Converter Manual (Tecan Document ID 396042).

http:\\www.tecan.com/HID


Section 1 Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup SystemOne-time tasks

Figure 1 Automated HID workflow for the Quantifiler® Duo kit. The Quantifiler Duo scripts, Converter software, and converted files are shown in red text and marked with *.

Create detectors for the 7500 Real-Time PCR System

If you have not already done so, create the following detectors in the SDS Software v1.2.3 for running the Quantifiler® Duo kit assays. Refer to the Applied Biosystems Quantifiler® Duo DNA Quantification Kit User’s Manual for details on creating the detectors.

IMPORTANT! The detectors must have the exact names and capitalization shown below.

Note: These detectors allow you to import the converted qPCR/STR system-generated 7500 setup file to the SDS Software.

Detector Name Reporter Dye Quencher Dye

Duo Human VIC Quencher Dye = (none) (make sure (none) is selected)

Duo Male FAM Quencher Dye = (none) (make sure (none) is selected)

Duo IPC NED Quencher Dye = (none) (make sure (none) is selected)


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwarePrepare qPCR reagents and labware

Prepare qPCR reagents and labware

Note: Perform routine maintenance before placing reagents, labware, and samples on the worktable. Refer to Chapter 2, “Pre-Run Procedures” in the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide.

Determine required reagent volumes

Note: Applied Biosystems recommends that you combine reagents from different tubes from the same lot if necessary to meet the minimum volume requirements.

1. Determine the volume of DNA standard and dilution buffer (or volume of pre-prepared DNA standard dilution series) that you need. You can either:

• Let the qPCR/STR system prepare the DNA standard dilution series for you. Use the table below to determine the required volume of undiluted Quantifiler® Duo DNA Standard and Quantifiler® Duo Dilution Buffer.

• Use a pre-prepared DNA standard dilution series, if the pre-prepared series meets the following three requirements:

– The series was prepared in the last two weeks.– The series was prepared according to the procedures specified in the

Quantifiler® Duo DNA Quantification Kit User’s Manual or was prepared by the qPCR/STR system.

– At least 30 µL of each of the eight concentrations of diluted standard is available. For reliable liquid detection, it is recommended to use at least 50 µL of each of the eight concentrations of diluted standard.

Note: Two replicates of the eight concentrations in the DNA standard dilution series are transferred to the qPCR reaction plate for a total of 16 DNA standard dilution series samples.30 µL includes the 4 µL necessary to prepare the two replicates of each concentration in the DNA standard dilution series, plus the minimum required overfill volume.

Required volumes for qPCR/STR system-prepared DNA standard dilution series

Reagent Available volume in full tube of reagent

Minimum volume‡ required on worktable

‡ Includes 50 µL dead volume per tube necessary to ensure that the pipette tips remain submerged during aspiration so that liquid, not air, enters the tips.

Undiluted Quantifiler® Duo DNA Standard

120 µL 75 µL

Quantifiler® Duo Dilution Buffer

1.8 mL 825 µL

7Q

uantifiler ® D

uo DN

A Quantification K

it Support and Validation – New

Scripts and Converter Softw

are

Section 1 Using the Quantifiler ®

Duo D

NA Quantification Kit w

ith the HID

EVOlution™

–qPCR/STR

Setup SystemPrepare qPCR

reagents and labware

2. Calculate the required volume of Quantifiler® Duo kit reagents using Table 2.

IMPORTANT! To obtain the required volume of primer mix, you can place up to 3 tubes of primer mix in the reagent block for one run. If you divide the minimum required volume of primer mix across 2 or 3 tubes, each tube must contain a multiple of 190 µL plus an additional 50 µL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions. Refer to “About minimum required reagent volumes” on page 25 of the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide.

Note: The volumes in the table include the excess volume required per sample and per run necessary to compensate for evaporation and pipetting losses during the run.

Table 2 Reagent volumes for qPCR reaction setup with the Quantifiler® Duo DNA Quantification Kit (25-µL reaction volume)

Reagent Available volume in

full tube of reagent

Required volume per

reaction

Extracted DNA

samples per run

Diluted standard samples per run

Required excess

reactions per run

Required dead

volume‡ per tube

‡ A 50-µL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid, not air, enters the tips.

Minimum required volume for 80 extracted DNA samples and

16 diluted standards =

[A×(B + C + D)] + E

A B C D E

Quantifiler® Duo Primer Mix

1.4 mL 11.55 µL up to 80 16 3 50 µL 1194§ µL in one tube#

§ [11.55 µL/reaction×(80 + 16 + 3 reactions)] + 50 µL/tube = 1193.45 µL in one tube.# If you divide the minimum volume of Primer Mix across multiple tubes, each tube must contain a multiple of 190 µL plus an additional 50 µL per tube to ensure that the instrument detects

adequate volume to prepare the selected number of reactions. For example, place at least 1000 µL in the first tube and 240 µL in the second tube.

Quantifiler® Duo PCR Reaction Mix

5 mL 13.75 µL up to 80 16 3 50 µL 1412‡‡ µL in one tube

‡‡[13.75 µL/reaction×(80 + 16 + 3 reactions)] + 50 µL/tube = 1411.25 µL in one tube.



Place reagents in the qPCR reagent block

Use the following procedure to place the Quantifiler® Duo kit reagents into the correct positions in the chilled qPCR reagent block.

1. Thaw the reagents, then prepare according to the Quantifiler® Duo DNA Quantification Kit User’s Manual:

• Thaw the primer mix completely, then vortex 3 to 5 seconds and centrifuge briefly before opening the tube.

• Swirl the PCR reaction mix gently before using. Do not vortex it.• Vortex the DNA standard 3 to 5 seconds.

2. If you are using a previously-prepared DNA standard dilution series, vortex and briefly centrifuge the DNA standard dilutions at low speed before use.

3. Place the labeled empty tubes (or labeled pre-prepared DNA standard dilution series) in positions 1 through 8 as shown in Table 3 on page 9.

IMPORTANT! Make sure to place each concentration in the correct location in the reagent block, as shown in the orange table in Table 3 on page 9.

4. Place the reagents in positions 9 through 15 as shown in Table 3 on page 9.


Section 1 Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup SystemPrepare qPCR reagents and labware

Table 3 Reagent block configuration for Quantifiler® Duo kit

Legend:

1-8. 1.5-mL tubes of pre-prepared DNA standard dilution series (arrange concentrations as shown in the corresponding orange table) OR 1.5-mL empty tubes for DNA standard dilution series.

9-11. Up to three 1.5-mL tube(s) of Quantifiler® Duo Primer Mix. Place the first tube in position 9. If you use more than one tube, continue to position 10, then position 11.‡

12. Quantifiler® Duo PCR Reaction mix.

13. Empty 5-mL VWR tube (VWR part number 890005-596) for the master mix.

14. (If the run includes preparation of DNA standard dilution series) Quantifiler® Duo DNA Standard tube.

15. (If the run includes preparation of DNA standard dilution series) Quantifiler® Duo Dilution Buffer tube.

‡ The required number of tubes with the Quantifiler® Duo Primer Mix depends on the number of reactions and the fill level of the tubes. The instrument aspirates 190 µL at a time. In order for the instrument to detect reagent in a tube, the tube must contain a minimum of 240 µL (190 µL plus a 50-µL dead volume). If the volume of reagent in the first tube (position 9) falls below 240 µL, the instrument looks for a sufficient volume in the position 10 tube, then in the position 11 tube. If the tubes are not available or the instrument does not detect sufficient volume, the instrument pauses the run and displays the error message “Not enough liquid.”



Set up the samples, reagents, and plasticware on the worktable

Set up the samples, reagents, and plasticware as described in Chapter 3 of the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide and shown below in Table 4.

Note: The TE buffer trough (7 in Table 4 below) is not used for Quantifiler Duo kit runs. Dilution buffer is provided with the kit and is placed in the reagent block.

Table 4 Tecan Freedom EVO® workstation (worktable) layout for a qPCR setup run

Note: Locations for DNA samples in both a plate and tubes are shown, but only one type of plasticware for extracted DNA can be placed on the workstation for a given run.

1–2. 200-µL disposable pipette tips (DiTis)

3–6. 50-µL DiTis

7. Not used with the Quantifiler® Duo kit: Trough for T10E0.1 buffer (used with Quantifiler® Human and Human Male DNA Quantification Kits only)

8. Chilled qPCR Reagent Block

9. MicroAmp® Optical 96-Well Reaction Plate (qPCR reaction plate) with 96-well metal plate adapter

10. MicroAmp® Optical 96-Well Reaction Plate (if extracted DNA samples are in a plate) with 96-well metal plate adapter

11. Tube racks S1 through S5 for DNA sample tubes (if extracted DNA samples are in tubes)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

81

2

3

4

5

6

7

9

10

11

S1 S5S2 S4S3

Site number 1

Site number 2

Site number 3

Grid numbers


Section 1 Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup SystemRun automated qPCR reaction setup

Run automated qPCR reaction setup

IMPORTANT! For all Quantifiler® kits, you can save time by creating a qPCR/STR Sample file to import sample information into the qPCR/STR system software. If you enter sample information manually for qPCR reaction setup, the information is not retained, and you will need to manually re-enter the information for STR PCR reaction setup. Refer to Chapter 2 of the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide for more information on qPCR/STR Sample files.

To run a script, follow the instructions in Chapter 4 of the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide using the appropriate Quantifiler® Duo script from the following table:

Perform qPCR amplification and review results

Convert and transfer the 7500 setup file

IMPORTANT! After qPCR Reaction setup (Quantifiler Duo kit only), you must use the HID EVOlution™ - Quantifiler® Duo Converter Software to convert the 7500 setup file (ReactionPlate1.txt or <barcode>.txt) generated during the qPCR reaction setup run. This creates an importable plate record containing run parameters (including detectors) needed to run the Quantifiler Duo kit assay on the 7500 instrument.

1. On the qPCR/STR system, navigate to the C:\HIDEVOlution_qPCRSTRfiles folder, then locate the file (ReactionPlate1.txt or <barcode>.txt).

2. (Optional) Rename the file.

3. Copy and transfer the ReactionPlate1.txt (or <barcode>.txt) file to a location where it can be accessed by the HID EVOlution™ - Quantifiler® Duo Converter Software.

Freedom EVOware® software script selection for qPCR reaction setup

If you are using the kit(s)...

and the sample DNA is in... ...use the script

Maximum number of extracted DNA samples per run

Quantifiler® Duo Kit a 96-well plate QuantifilerDuo_plate.esc 80

1.5-mL microcentrifuge tubes

QuantifilerDuo_tubes.esc 80


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwarePerform qPCR amplification and review results

4. Convert the file:

a. Start the Converter.

b. Select Convert Quantifiler Duo 7500 Setup file, then follow the prompts in the software.

Note: Refer to the HID EVOlution™ - qPCR/STR Setup System, HID EVOlution™ - Combination System, Quantifiler® Duo Converter Manual (Tecan Document ID 396042) for details.

5. Copy and transfer the converted 7500 setup file to a location where it can be accessed by the SDS software computer.

Import the converted 7500 setup file and perform qPCR

1. Start the 7500 Real-Time PCR System and SDS Software:

a. Start the computer.

b. Power on the 7500 instrument.

c. Start the SDS Software.

Note: For more information on starting the 7500 Real-Time PCR System and software, refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide.

2. Load the qPCR reaction plate into the 7500 instrument (refer to the Quantifiler® Duo DNA Quantification Kit User’s Guide for details.)

3. Import the converted 7500 setup file into the Applied Biosystems 7500 SDS Software plate document for use during the quantitation run as follows:

a. In the SDS software, select File New to create a new Absolute Quantification (Standard Curve) plate document, then in the new document wizard select Finish.

b. With a blank plate document open, select File Import Sample Setup.

c. Browse to locate the converted 7500 setup file, select the file, then click Open.

d. In the plate document, select the Instrument tab, then:• Change the Sample Volume to 25 (µL)• Select the 9600 Emulation box

Note: The thermal cycler protocol validated for use with the Quantifiler® Duo kit includes a hold step at 50 °C for 2 minutes.

e. Omit any blank wells.


Section 1 Using the Quantifiler® Duo DNA Quantification Kit with the HID EVOlution™ – qPCR/STR Setup SystemPerform qPCR amplification and review results

4. Save the plate document:

a. Select File Save As.

b. In the Save as dialog box:• Enter a file name in the File name field. Do not add an extension to the

file name.• Select SDS Document (*.sds) from the Save as type drop-down list.• Click Save.

5. Select the Instrument tab, then click Start.

Analyze and review the qPCR results

1. In the SDS software, click Analyze.

2. Review the standard curve to ensure that results are within the typical range (refer to the Quantifiler® Duo DNA Quantification Kit User’s Manual). If necessary, omit standard outliers, then click Analyze to recalculate the standard curve.

3. Review the quantitation data and make the necessary determinations:

a. Review the quantitation data, including the IPC CT and the ratio of male to female DNA, and consider the original condition of the sample.

b. For each sample, determine if:• The sample requires additional processing before STR PCR reaction

setup.• You will proceed with autosomal STR or Y STR reaction setup.

IMPORTANT! The qPCR/STR system sets up STR PCR reactions with one AmpFlSTR® kit per run. We recommend that you view your qPCR results in the SDS software, then determine how you will batch samples for amplification with the appropriate AmpFlSTR kit.

• You need to adjust the target input amount to add more or less DNA to the amplification reaction.

Note: After you import the qPCR results to the qPCR/STR system software, you can adjust the target input amount in the Sample Normalization Adjustment window.

Note: Refer to “Interpretation of Results” in the Quantifiler® Duo DNA Quantification Kit User’s Guide for details.


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwarePerform qPCR amplification and review results

Export and convert the 7500 results files

Note: During STR PCR reaction setup, you import the converted 7500 results files to the qPCR/STR system. The EVOware uses the converted 7500 results file to normalize the DNA input amount for STR PCR reaction setup on the qPCR/STR system.

When you are done analyzing the results in the SDS software and have determined the appropriate AmpFlSTR® kit for use with each sample, export the 7500 results file(s), convert the file(s), then copy them to the qPCR/STR system computer:

1. In the SDS software, select File Export Results.

2. In Save as type, select Results Export Files (*.csv), then click Save.

3. Use the Converter to create two results files (a human file and a male file).

Note: Refer to the HID EVOlution™ - qPCR/STR Setup System, HID EVOlution™ - Combination System, Quantifiler® Duo Converter Manual (Tecan Document ID 396042) for details.

4. Copy the converted results files to the computer where the Freedom EVOware® software is installed, or to a server that can be accessed from both systems.

SECTION 1

15Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter Software

STR PCR reaction setup

When performing STR PCR reaction setup using samples quantified with the Quantifiler Duo kit:

1. Prepare STR PCR reagents and labware as described in Chapter 6 of the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide.

2. Run STR PCR setup as described in Chapter 7 of the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide, except that you “Import the converted 7500 results files” as described below.

Import the converted 7500 results files

IMPORTANT! The qPCR/STR system sets up STR PCR reactions with one AmpFlSTR® kit per run. We recommend that you view your qPCR results in the SDS software, then determine how you will batch samples for amplification with the appropriate AmpFlSTR kit.

1. When you are prompted to import the 7500 results, import either the converted human or male 7500 results files based on the AmpFlSTR® kit that you selected for use with the samples.

2. Review the information in the Sample Normalization Adjustment window:

a. If necessary, adjust the Req. Amt[ng] to increase or decrease the target DNA input amount added during reaction setup.

b. If you want to include samples for STR setup that are not automatically selected:

• If necessary, adjust the Req. Amt[ng] to increase or decrease the target DNA input amount added during reaction setup.

• Select the Process check box.

Note: For samples with undetermined quantification results in the imported 7500 results file(s), the system adds the maximum volume of DNA sample per reaction. You cannot increase or decrease the volume of DNA sample added to the reaction by changing the value in the Req. Amt.[ng] column.


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwareSTR PCR reaction setup

Section 2 Validation of Quantifiler® Duo Kit on HID EVOlution™ – qPCR/STR Setup System

This section covers:

■ Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

■ Experiments and results: Freedom EVOware® Software v1.4 . . . . . . . . . . . . . . . . 21

■ Experiments and results: Freedom EVOware® Software v2.1 . . . . . . . . . . . . . . . . 28

■ Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

SECTION 2

17Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter Software

Overview

The Quantifiler® Duo DNA Quantification Kit (Quantifiler® Duo kit) is now supported on the HID EVOlution™ – qPCR/STR Setup System (qPCR/STR system).

This section describes the validation and verification studies of quantitative PCR (qPCR) setup on the qPCR/STR system using the new Quantifiler Duo scripts and the Quantifiler Duo Converter Software v1.0 that support the Quantifiler® Duo kit.

The following studies were performed:

• Precision and reproducibility studies to evaluate the ability to reproducibly set up Quantifiler Duo reactions using the Quantifiler Duo scripts (EVOware® software v1.4 and v2.1)

• Contamination studies to evaluate the potential for cross-contamination (EVOware® software v1.4 and v2.1)

• Verification studies to evaluate that all files converted using the Quantifiler Duo Converter are compatible with the 7500 Software v1.2.3 and the HID EVOlution system (normalization and STR setup) and that all sample information is complete and accurate (EVOware® software v1.4)


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwareMaterials and methods

Materials and methods

Materials

Component Description

Converter HID EVOlution™ Quantifiler® Duo Converter v1.0.0.0

qPCR/STR system • Freedom EVO® 150 robotic workstation• 4-channel LiHa• Freedom EVOware® software (see “Experiments and results: Freedom EVOware®

Software v1.4” on page 21 or “Experiments and results: Freedom EVOware® Software v2.1” on page 28 for studies performed on software versions):– v1.4 SP1, Tecan HID EVOlution driver 1.0.0.24, and Tecan Sample Oriented

EVOware Limited Edition SP1 or

– v2.1, Tecan HID EVOlution driver 1.0.0.25 SP4, Tecan Sample Oriented EVOware 1.2 SP1

• Windows XP Professional operating system• New qPCR setup scripts:

– QuantifilerDuo_plate– QuantifilerDuo_tubes

Instruments and software • 3130xl Genetic Analyzer Data Collection Software v3.0• GeneMapper® ID-X Software version 1.0• GeneAmp® PCR System 9700 Thermal Cycler gold-plated silver 96-well block• 7500 Real-Time PCR System with SDS Software v 1.2.3

Consumables • 50 µL and 200 µL tips (Tecan Group Ltd.) • TE buffer (Teknova, Hollister, CA)

Chemistry Kits Quantifiler® Duo DNA Quantification Kit (PN 4387746/Lot Number 0905008)

AmpFlSTR® Identifiler® PCR Amplification Kit (PN 4322288/Lot Number 0809108)

AmpFlSTR® Yfiler® PCR Amplification Kit (PN 4359513/Lot Number 0904046)

AmpFlSTR® MiniFiler™ PCR Amplification Kit (PN 4373872/Lot Number 0904019)

Sample DNA 1 female sample (anonymous donor, Serological Research Institute, S223, L/N 0909240)

1 male sample (Biochain, Hayward, CA)


Section 2 Validation of Quantifiler® Duo Kit on HID EVOlution™ – qPCR/STR Setup SystemMaterials and methods

Extracted DNA sample plate and tubes set up

The DNA concentrations in one anonymous female (Serological Research Institute) and one commercial male genomic DNA sample (Biochain Institute) were determined using the Quantifiler® Duo DNA Quantification Kit by manual setup and in triplicate.

The samples were then serially diluted manually to obtain concentrations of 50, 10, 5, 2, 1, 0.5, 0.1, and 0.025 ng/µL.

One sample plate containing extracted DNA (source plate) and one set of 1.5 mL tubes containing extracted DNA (source tubes) were prepared manually with 2 to 4 replicates for each concentration and TE buffer blanks (Figures 2 and 3).

Figure 2 Sample plate set up with extracted DNA (source plate)

Figure 3 Sample tubes (1.5 mL) set up with extracted DNA (source tubes)


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwareMaterials and methods

qPCR reaction plate setup

The following scripts, worktable layouts, and Quantifiler Duo reagent block configuration were used.

The source plate (Figure 2 on page 19) and the source tubes (Figure 3 on page 19) were then run on each system on three consecutive days to create six qPCR reaction plates per system (Figure 4).

During each qPCR reaction setup run, a fresh standard dilution series of the Quantifiler Duo DNA Standard was prepared on the qPCR/STR system.

Figure 4 Precision and reproducibility studies – qPCR reaction plate setup runs

System Setup Script Worktable layout Reagent block configuration

qPCR/STR system qPCR • QuantifilerDuo_plate• QuantifilerDuo_tubes

See Table 4 on page 10

See Table 3 on page 9


Section 2 Validation of Quantifiler® Duo Kit on HID EVOlution™ – qPCR/STR Setup SystemExperiments and results: Freedom EVOware® Software v1.4

Studies

Precision and reproducibility studies

On three consecutive days, the two qPCR reaction plates generated from each qPCR setup run (Figure 4 on page 20) were run on the 7500 Real-Time PCR system (six runs total per system). Standard curve slope, R2, and sample DNA concentration ranges were evaluated for the Ribonuclease P RNA Component H1 target (RPPH1, total human) and Sex-determining Region Y target (SRY, male).

Contamination studies

CT values from the precision and reproducibility studies were evaluated. Female samples with CT values <40 for the Quantifiler Duo Male target and TE buffer blanks with CT values <40 were manually set up for amplification using the MiniFiler™ PCR Amplification Kit or the Yfiler® PCR Amplification Kit. STR profiles were evaluated in the GeneMapper ID-X software.

Normalization table verification studies

The 7500 results files generated from Plate runs 1, 2, and 3 and Tubes runs 1, 2, and 3 from each system were processed with the Quantifiler Duo Converter to generate human and male 7500 results files. The converted human and male results files from each system were imported into the appropriate Quantifiler Duo script. The dilution schemes (shown in the script Sample Normalization Adjustment window) that the scripts generated for each sample were evaluated.

Experiments and results: Freedom EVOware® Software v1.4

Precision and reproducibility studies

Experiment On three consecutive days, the two qPCR reaction plates generated from each qPCR setup run (Figure 4 on page 20) were run on the 7500 Real-Time PCR system (six runs total). Standard curve slope, R2, and sample DNA concentration values were evaluated for the Ribonuclease P RNA Component H1 target (RPPH1, total human) and Sex-determining Region Y target (SRY, male)

Slope and R2 Observed slope and R2 values are summarized in Table 5. The slope and R2 values were within the typical range of slope values observed during the development and validation of the Quantifiler® Duo kit (slope: –3.0 to –3.6 and R2: >0.98, respectively).

Table 5 Slope and R2 values for standard curves

Run Source plate Source tubes

Duo Human Duo Male Duo Human Duo Male

Slope R2 Slope R2 Slope R2 Slope R2

1 –3.02 0.997 –3.18 0.998 –3.28 0.998 –3.42 0.995

2 –3.19 0.998 –3.22 0.996 –3.24 0.997 –3.37 0.994

3 –3.24 0.998 –3.39 0.992 –3.29 0.998 –3.39 0.997

Avg –3.15 0.998 –3.26 0.995 –3.27 0.998 –3.39 0.995


Quantifiler® Duo DNA Quantification Kit Support and Validation – New Scripts and Converter SoftwareExperiments and results: Freedom EVOware® Software v1.4

DNA concentration The range of variation in the quantification results is:

• Comparable to the results seen during developmental validation (reported in the Quantifiler® Duo PCR Amplification User Guide) at all tested concentrations of DNA (among replicates and across three runs)

• Similar for source plate and source tubes

DNA concentrations from the three runs for the source plate and tubes are summarized in Figures 5 through 8 on pages 22 and 24.

The standard deviation values and concentration ranges are summarized in Table 6 on page 25 and Table 7 on page 26.

Figure 5 Quantification results obtained from the RPPH1 target (total human) qPCR reaction plates prepared using QuantiflerDuo_plate script from DNA source plate



Figure 6 Quantification results obtained from the SRY target (male) in the qPCR reaction plates prepared using QuantiflerDuo_plate script from DNA source plate

Figure 7 Quantification results obtained from the RPPH1 target (total human) in the qPCR reaction plates prepared using QuantiflerDuo_tubes script from DNA source tubes



Figure 8 Quantification results obtained from the SRY target (male) in the qPCR reaction plates prepared using QuantiflerDuo_tubes script from DNA source tubes



Table 6 RPPH1 target (human) DNA standard deviations and concentrations

Male Sample

Standard Deviations

Expected (ng/µL)

n (for

Plate)

Plate 1 Plate 2 Plate 3 n (for

Tubes)

Tubes 1 Tubes 2 Tubes 3

0.025 4 0.009 0.022 0.010 4 0.028 0.009 0.005

0.1 4 0.009 0.024 0.021 2‡

‡ Due to the number of wells on the qPCR reaction plate, only two samples were run. Standard deviation not calculated.

N/A‡ N/A‡ N/A‡

0.5 4 0.018 0.008 0.052 4 0.025 0.018 0.052

1 4 0.033 0.023 0.069 4 0.066 0.123 0.147

2 4 0.079 0.133 0.162 4 0.048 0.192 0.255

5 4 0.351 0.235 0.331 4 0.595 0.574 0.656

10 4 0.311 0.843 0.849 4 0.598 0.541 0.346

50 4 3.949 1.505 1.488 4 0.888 1.386 3.261

Female Sample

Concentration (ng/µL)

(Expected ng/µL)

n (for Plate)

Plate 1 Plate 2 Plate 3 n (for Tubes)


0.5 2 0.35; 0.32 0.31; 0.32 0.45; 0.46 2 0.46; 0.42 0.46; 0.46 0.60; 0.55

1 2 0.76; 0.67 0.64; 0.63 0.92; 0.85 2 0.99; 0.86 0.98; 0.89 1.08; 1.12

2 2 1.53; 1.54 1.19; 1.46 1.85; 1.87 2 1.82; 1.82 1.63; 1.84 2.05; 2.04

5 2 4.12; 4.50 3.44; 3.86 4.88; 4.94 2 4.37; 4.55 4.66; 4.38 5.43; 5.45

10 2 8.98; 9.92 7.05; 7.70 9.63; 10.15

2 9.48; 10.74

10.59; 10.48

12.32; 11.85

50 2 59.86; 64.48

42.85; 46.47

53.61; 54.78

2 54.92; 58.79

57.46; 58.41

64.06; 65.04



Table 7 SRY target (male) DNA standard deviations

Contamination study

Experiment CT values from the precision and reproducibility studies were evaluated.

Results The results of the contamination study are summarized in Table 8.

Five TE buffer blanks exhibited CT values <40 and were manually set up for amplification using the MiniFiler™ PCR Amplification Kit. None of these 5 TE buffer blanks exhibited STR profiles.

One female sample exhibited a CT value of 37.97 for the Duo Male target and was manually set up for amplification using the Yfiler® and MiniFiler™ PCR Amplification kits. However, this sample did not yield a Y-STR profile when amplified using the Yfiler PCR Amplification Kit. Similarly, the autosomal STR profile obtained using the MiniFiler kit did not indicate the presence of a mixture. Therefore, the lower CT values are attributed to nonspecific amplification.

Male Sample

Standard Deviations

Expected (ng/µL)

n (for

Plate)

Plate 1 Plate 2 Plate 3 n (for

Tubes)


0.025 4 0.007 0.019 0.023 4 0.011 0.011 0.013

0.1 4 0.010 0.012 0.022 2‡


N/A‡ N/A‡ N/A‡

0.5 4 0.029 0.016 0.031 4 0.039 0.021 0.066

1 4 0.037 0.063 0.066 4 0.059 0.091 0.180

2 4 0.141 0.041 0.107 4 0.090 0.133 0.289

5 4 0.263 0.399 0.448 4 0.425 0.624 0.873

10 4 0.275 0.601 0.958 4 0.738 0.737 1.188

50 4 5.385 0.948 0.615 4 5.619 1.597 2.549



Table 8 TE buffer blank CT and STR results (N/A = samples not set up for amplification)

Verification of normalization setup

Experiment The 7500 results files generated from Plate runs 1, 2, and 3 and Tubes runs 1, 2, and 3 from each system were processed with the Quantifiler Duo converter to generate human and male 7500 results files. The converted human and male results files from each system were imported into the appropriate Quantifiler Duo script. The dilution schemes (shown in the script Sample Normalization Adjustment window) that the scripts generated for each sample were evaluated.

Normalization values

The dilution schemes (shown in the script Sample Normalization Adjustment window) that the scripts generated for each sample were evaluated. Total dilution (T Dil.) values for each concentration of human and male DNA were examined to ensure that the calculated dilutions were appropriate for each concentration. Refer to the HID EVOlution™ – qPCR/STR Setup System Getting Started Guide for information on the dilution protocols used by the qPCR/STR system software.

Run Total # Samples

Female samples with

CT <40 for male detector

STR Results Total # TE

blanks

TE Blanks Exhibiting CT <40 for male and/or

human detector

STR Results

Yfiler Kit MiniFiler Kit

MiniFiler Kit

Plate 1 44 None N/A N/A 36 None N/A

Plate 2 44 None N/A N/A 36 TEBlank 5 (well A2): Duo Human CT = 37.03

No STR profile

Plate 3 44 None N/A N/A 36 TEBlank 17 (well B5): Duo Male CT = 37.65

No STR profile

TEBlank 20 (well H5): Duo Human CT = 39.20

No STR profile

Tube 1 42 None N/A N/A 38 None N/A

Tube 2 42 None N/A N/A 38 TEBlank 3 (position 6, carrier 1): Duo Male CT = 38.20

No STR profile

Tube 3 42 2 ng/µL female sample (position 29 in carrier 2) exhibited CT value of 37.97 for male target

No Male STR profile was detected with Yfiler

No indication of mixture STR profile

38 TEBlank 15 (position 30, carrier 2): Duo Human CT = 36.85

No STR profile



Experiments and results: Freedom EVOware® Software v2.1

Precision and reproducibility study

Experiment The source plate (Figure 2 on page 19) and source tubes (Figure 3 on page 19) were run using the QuantifilerDuo_plate script. The two qPCR reaction plates were run on the 7500 Real-Time PCR system. Sample DNA concentration were evaluated.

DNA concentration The range of variation in the quantification results is:

• Comparable to the results seen during developmental validation (reported in the Quantifiler® Duo PCR Amplification User Guide) at all tested concentrations of DNA.

• Similar for source plate and source tubes.

DNA concentrations are summarized in Figures 9 through12 on pages 29 and page 30.

The standard deviation values and concentration ranges for human and male DNA quantity values are summarized in Tables 9 and 10 on page 31.



Figure 9 EVOware® software v2.1: Quantification results obtained from the RPPH1 target (total human) qPCR reaction plates prepared using QuantiflerDuo_plate script from DNA source plate

Figure 10 EVOware® software v2.1: Quantification results obtained from the SRY target (male) in the qPCR reaction plates prepared using QuantiflerDuo_plate script from DNA source plate



Figure 11 EVOware® software v2.1: Quantification results obtained from the RPPH1 target (total human) in the qPCR reaction plates prepared using QuantiflerDuo_tubes script from DNA source tubes

Figure 12 EVOware® software v2.1: Quantification results obtained from the SRY target (male) in the qPCR reaction plates prepared using QuantiflerDuo_tubes script from DNA source tubes



Table 9 RPPH1 target (human) DNA standard deviations and concentrations

Table 10 SRY target (male) DNA standard deviations and concentrations

Male Sample Standard Deviations

Expected (ng/µL)

n (for Plate)

Plate n (for Tubes)

Tubes

0.025 4 0.008 4 0.007

0.1 4 0.024 2‡


N/A‡

0.5 4 0.080 4 0.054

1 4 0.161 4 0.183

2 4 0.354 4 0.120

5 4 0.450 4 0.324

10 4 0.992 4 0.319

50 4 7.082 4 5.746

Female Sample

Concentration (ng/µL)

Expected (ng/µL)

n (for Plate)

Plate n (for Tubes) Tubes

0.5 2 0.59; 0.71 2 0.61; 0.54

1 2 1.29; 1.35 2 1.10; 1.18

2 2 2.52; 2.59 2 2.05; 1.93

5 2 7.73; 7.27 2 6.12; 5.17

10 2 14.70; 14.84 2 12.52; 12.52

50 2 90.35; 83.46 2 79.98; 75.85

Male Sample

Standard Deviations

Expected (ng/µL)

n (for

Plate)

Plate n (for Tubes)

Tubes

0.025 4 0.015 4 0.011

0.1 4 0.044 2 N/A

0.5 4 0.053 4 0.056

1 4 0.243 4 0.179

2 4 0.512 4 0.130

5 4 0.645 4 0.518

10 4 1.223 4 0.928

50 4 3.328 4 2.727



Contamination study

Experiment CT values from the precision and reproducibility studies were evaluated. If any female samples exhibited CT values <40 for the Quantifiler Duo Male target and if any TE buffer blanks exhibited CT values <40, they were manually set up for amplification using the MiniFiler™ PCR Amplification Kit or the Yfiler® PCR Amplification Kit. STR profiles were evaluated in the GeneMapper ID-X software.

Results The results of contamination study are summarized in Table 11.

One TE buffer blank exhibited CT value <40 and was manually set up for amplification using the MiniFiler™ PCR Amplification Kit. However, this TE buffer blank did not exhibit an STR profile when amplified using the MiniFiler™ kit. Therefore, the lower CT value is attributed to nonspecific amplification.

Table 11 TE buffer blank CT and STR results (N/A = samples not set up for amplification)

Run # Samp

# of Samples with CT<40

STR Results# TE Blank

# TE Blanks Exhibiting CT <40

STR Results

Yfiler Kit MiniFiler Kit

MiniFiler Kit

Plate 44 None N/A N/A 36 None N/A

Tube 42 None N/A N/A 38 TEBlank24 (position 47, carrier 3): Duo Human Ct is 38.84

No STR profile


Section 2 Validation of Quantifiler® Duo Kit on HID EVOlution™ – qPCR/STR Setup SystemConclusions

Conclusions

The Quantifiler Duo scripts and Quantifiler Duo converter were validated on the HID EVOlution™ – qPCR/STR Setup System using the Quantifiler® Duo DNA Quantification Kit:

• Precision and reproducibility studies confirm that the new Quantifiler Duo scripts and Quantifiler Duo converter provide reliable results at different DNA input amounts on Freedom EVOware® Software v1.4 and v2.1.

• Contamination studies demonstrated clean liquid handling capabilities for the qPCR setup on EVOware® Software v1.4 and v2.1.

• Normalization and STR setup studies confirm that the new Quantifiler Duo scripts and Quantifiler Duo converter provide accurate and reliable data handling for normalization and STR setup.

• The stability study confirms that the Quantifiler Duo scripts prepare diluted standards that meet the performance requirements when stored according to the conditions recommended in the Quantifiler® Duo DNA Quantification Kit User Guide.

Part Number 4445275 Rev. B 09/2010 Stock Number 112UB29-01

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HID EVOlution – qPCR/STR Setup System · USER BULLETIN | SEPTEMBER 2010 HID EVOlution™ –...

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Transcript of HID EVOlution – qPCR/STR Setup System · USER BULLETIN | SEPTEMBER 2010 HID EVOlution™ –...